The Reliability of Estimating Ki Values for Direct, Reversible Inhibition Of

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Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2015/09/09/dmd.115.066597.DC1

1521-009X/43/11/1744–1750$25.00 http://dx.doi.org/10.1124/dmd.115.066597 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1744–1750, November 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments s

Lois J. Haupt, Faraz Kazmi, Brian W. Ogilvie, David B. Buckley, Brian D. Smith, Sarah Leatherman, Brandy Paris, Oliver Parkinson, and Andrew Parkinson

XenoTech, LLC, Lenexa, Kansas (L.J.H., F.K., B.W.O., D.B.B., B.D.S., S.L.); and XPD Consulting, Shawnee, Kansas (B.P., O.P., A.P.)

Received July 28, 2015; accepted September 8, 2015

ABSTRACT

In the present study, we conducted a retrospective analysis of 343 in low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Downloaded from vitro experiments to ascertain whether observed (experimentally Under these conditions, predicted Ki values, based on IC50/2, corre- determined) values of Ki for reversible cytochrome P450 (P450) lated strongly with experimentally observed Ki determinations [r = inhibition could be reliably predicted by dividing the corresponding 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values,

IC50 values by two, based on the relationship (for competitive in- 316 (92%) were within a factor of 2 of the experimentally determined Ki hibition) in which Ki =IC50/2 when [S] (substrate concentration) = Km values, and only one value fell outside a 3-fold range. In the case of

(Michaelis-Menten constant). Values of Ki and IC50 were determined noncompetitive inhibitors, Ki values predicted from IC50/2 values were dmd.aspetjournals.org under the following conditions: 1) the concentration of P450 marker overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be substrate, [S], was equal to Km (for IC50 determinations) and spanned expected because, for noncompetitive inhibition, Ki =IC50 (not IC50/2).

Km (for Ki determinations); 2) the substrate incubation time was short The results suggest that, under appropriate experimental conditions

(5 minutes) to minimize metabolism-dependent inhibition and inhibitor with the substrate concentration equal to Km, values of Ki for direct, depletion; and 3) the concentration of human liver microsomes was reversible inhibition can be reliably estimated from values of IC50/2. at ASPET Journals on September 28, 2021

Introduction the P450 enzyme inhibited by the investigational drug (i.e., fractional When an investigational drug is evaluated in vitro as a direct, metabolism by an enzyme, or fm = 1.0) (Ito et al., 1998; Rodrigues reversible inhibitor of human liver cytochrome P450 (P450) enzymes et al., 2001). When other pathways contribute to clearance of the probe , based on the Food and Drug Administration’s (FDA’s) 2012 draft drug, such that fm 1, the fold increase in plasma AUC of the probe Guidance for Industry on drug interactions (http://www.fda.gov/ drug in the presence of the inhibitory drug is given by eq. 2: downloads/Drugs/GuidanceComplianceRegulatoryInformation/ 1 AUCR ¼ ð2Þ Guidances/UCM292362.pdf), the need for a clinical drug interaction 1 × × þ 2 × ½I fm fm:enzyme 1 fm fm:enzyme 1þ study is based on eq. 1: Ki;unbound

½I A clinical drug interaction study is recommended for P450 en- AUCR ¼ R1 ¼ 1 þ ð1Þ Ki;unbound zymes other than CYP3A when the ratio R1 . 1.1 (where R1 5 1 1 [I]/Ki,unbound). The same criteria apply to significant circulating meta- where [I] is the maximum total (bound + unbound) plasma bolites, defined by the FDA as metabolites whose plasma AUC is $25% concentration of drug at steady state (Cmax,ss), and Ki,unbound is the of the parent AUC following dosing to steady state with the maximum dissociation constant for the enzyme-inhibitor complex for direct, clinical dose. The European Medicines Agency’s(EMA’s) 2012 reversible inhibition based on the concentration of unbound drug in the Guideline on the Investigation of Drug Interactions (http://www.ema. in vitro test system. AUCR, the area under the plasma concentration- europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/ time curve (AUC) ratio, represents the fold increase in plasma AUC of WC500129606.pdf) is based on a similar type of ratio and cutoff value, a probe drug whose clearance is entirely determined by metabolism by namely, [I]unbound/Ki,unbound $ 0.02, where Ki,unbound is as defined earlier and [I]unbound is the unbound (free) maximum concentration of drug in plasma (mean Cmax obtained following treatment with the highest Parts of this work were previously presented at the following meeting: Haupt L, recommended clinical dose). The EMA’s guideline also applies to Kazmi F, Ogilvie B, Buckley D, Smith B, Leatherman S, and Parkinson A (2011) significant circulating metabolites, defined as phase 1 metabolites whose Can Ki values for direct inhibition of CYP enzymes be reliably estimated from IC50 . . values? Seventeenth North American Regional ISSX Meeting; 2011 Oct 16–20; plasma AUC is 25% of the parent drug and 10% of total circulating Atlanta, GA. drug-derived material. To evaluate the potential for reversible inhibition dx.doi.org/10.1124/dmd.115.066597. of CYP3A enzymes in the intestine, the concentration of inhibitor in eq. 1 s This article has supplemental material available at dmd.aspetjournals.org. is defined as [I]gut = molar dose/250 ml. Values of [I]gut can greatly

ABBREVIATIONS: AFE, average fold error; AUC, area under the plasma concentration-time curve; EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration; NRMSE, normalized root mean square error; P450, cytochrome P450; RMSE, root mean square error.

1744 Estimating Ki from IC50 Values for P450 Inhibition 1745 exceed values of [I] (total Cmax,ss); accordingly, the FDA’s cutoff value for (internal standard), and dehydronifedipine-d6 (internal standard) were purchased inhibition of intestinal P450 enzymes is 11 (based on 1 + [I]gut/Ki,unbound). from SynFine Research (Richmond Hill, Ontario, Canada). 10-Deacetyltaxol (internal standard) was purchased from A.F. Hauser, Inc. Pharmaceutical The corresponding EMA cutoff is 10 (based on [I]gut/Ki,unbound). Typically, inhibition of P450 enzymes is first evaluated in vitro by (Valparaiso, IN). Investigational Drugs. determining the concentration of investigational drug (or significant A total of 132 investigational drugs and drug metabolites were examined during the course of P450 inhibition studies circulating metabolite) that causes 50% inhibition of P450 enzyme sponsored by numerous pharmaceutical companies in the United States, Europe, activity (IC50) with a selective P450 probe at a substrate concentration and Japan. Unfortunately, because they are proprietary compounds and for approximately equal to Km (Michaelis-Menten constant). Determining reasons of confidentiality, we are not at liberty to disclose the identity of the the mechanism of reversible inhibition (competitive, noncompetitive, structures of these compounds. The compounds represent a set of structurally mixed, or uncompetitive) and measuring the value of Ki requires an in diverse, small drug molecules under development for several different thera- vitro evaluation of the effects of multiple inhibitor concentrations peutic indications. The design of the experiments and the interpretation of the versus multiple substrate concentrations, ideally with the former results of this study (a comparison of two endpoints of P450 inhibition) required neither knowledge of chemical structures nor physicochemical properties. spanning Ki and the latter spanning Km. Determining values of IC50 and Test System. Pooled human liver microsomes (n = 16 or 200; mixed gender) Ki based on the unbound concentration of inhibitor in the test system (typically human liver microsomes) requires knowledge of fu (the were prepared from nontransplantable livers and characterized at XenoTech, inc LLC (Lenexa, KS) as described previously (Pearce et al., 1996; Parkinson et al., fraction of unbound drug in the microsomal incubation), which can be 2004). determined experimentally or estimated theoretically from the inhib-

Incubation Conditions. K and IC values were determined in accordance Downloaded from ’ i 50 itor s logP or logD value and the concentration of microsomal protein with recommendations in the FDA and EMA guidance documents and consensus (Austin et al.., 2002; Hallifax and Houston, 2006). papers (Tucker et al., 2001; Bjornsson et al., 2003). All experiments were The relationship between Ki and values of IC50 determined when [S] performed under the following conditions: 1) the concentration of P450 marker (substrate concentration) = Km depends on the mechanism of inhibition, substrate was approximately equal to Km for IC50 determinations and spanned as summarized in Table 1 (Cheng and Prusoff, 1973; Brandt et al., Km for Ki determinations (i.e., [S] ranged from 0.25 times Km to 10 times Km, solubility permitting); 2) the substrate incubation time was 5 minutes to minimize 1987; Cer et al., 2009). In the case of noncompetitive inhibition, Ki = metabolism-dependent inhibition and inhibitor depletion; and 3) the concentra- dmd.aspetjournals.org IC50. In the case of competitive and uncompetitive inhibition, Ki =IC50/ 2. In the case of mixed inhibition, K values range from IC to IC /2. tion of human liver microsomes was 0.1 mg/ml or less to maximize the unbound i 50 50 inhibitor concentration. The FDA’s 2012 Guidance for Industry on drug interactions acknowl- In general, incubations were conducted at 37C in 200- or 400-ml incubation edges that Ki values are often estimated from values of IC50/2, based on mixtures containing potassium phosphate buffer (50 mM, pH 7.4), MgCl2 the conservative assumption that the mechanism of reversible inhibition (3 mM), EDTA (1 mM), NADPH-generating system, and human liver microsomes. is competitive in nature. In the present study, we conducted a retro- Most of the P450 reactions examined have been described in detail elsewhere spective analysis of 343 in vitro Ki determinations to investigate (Paris et al., 2009; Parkinson et al., 2011). The P450 substrates, analytes whether experimentally determined Ki values can, in fact, be reliably (metabolites measured), internal standards, and microsomal protein concentra- at ASPET Journals on September 28, 2021 estimated from values of IC50/2 when IC50 values are determined under tion for all of the P450 reactions examined are summarized in Supplemental Table 1. conditions of [S] Km. Analytical Methods. All metabolites and their internal standards (usually isotopically labeled metabolites) were measured with validated liquid Materials and Methods chromatography–tandem mass spectrometry methods on AB Sciex (Framingham, Chemicals and Reagents. The commercial sources of most substrates, MA) API 2000, 3000, or 4000 mass spectrometers with Shimadzu (Kyoto, metabolites, internal standards, and reagents have been described previously Japan) high-performance liquid chromatography pumps and autosampler (Pearce et al., 1996; Paris et al., 2009; Parkinson et al., 2011). Efavirenz was systems according to methods described previously (Paris et al., 2009; Parkinson purchased from U.S. Pharmacopeia (Rockville, MD). 8-Hydroxyefavirenz, et al., 2011). Peak areas for all metabolites were integrated with an AB Sciex 8-hydroxyefavirenz-d4 (internal standard), and 6a-hydroxypaclitaxel-d5 (internal Analyst data system, and metabolites were quantified by reference to a standard standard) were purchased from Toronto Research Chemicals, Inc. (North York, calibration curve based on back-calculation of a weighted (1/x), linear, least- Ontario, Canada). The following chemicals were purchased from Sigma-Aldrich squares regression. 9 (St. Louis, MO): coumarin, paclitaxel, bufuralol, 1 -hydroxybufuralol, chlor- Data Processing. IC50 data were processed with one of two validated zoxazone, 6-hydroxychlorzoxazone, 6-hydroxychlorzoxazone-d2 (internal stan- software packages, Galileo- Laboratory Information Management System dard), nifedipine, and oxidized nifedipine. 7-Hydroxycoumarin was purchased (Galileo version 3.3; Thermo Fisher Scientific Inc., Grand Island, NY) or XLfit from Cerilliant (Round Rock, TX). 6a-Hydroxypaclitaxel, 7-hydroxycoumarin-d5 (version 3.0.5; ID Business Solutions Ltd., Guildford, Surrey, UK), which is

TABLE 1

Relationship between Ki and IC50 (when [S] = Km) for different mechanisms of reversible enzyme inhibition (adapted from Cheng and Prusoff, 1973; Brandt et al., 1987; Cer et al., 2009)

Type of Inhibition Modified Michaelis-Menten Equationa Relationship between Ki and IC50 when [S] = Km × ½ ¼ Vmax S ¼ IC50 Competitive v Ki 2 ½I Km 1þ þ½S Kia × ½ ¼ Vmax S ¼ IC50 Uncompetitive v Ki 2 ½I Km þ½S × 1þ Kib × ½ ¼ Vmax S ¼ IC50 Mixed v Ki IC50 to 2 depending on the ratio of Kia to Kib ½I ½I Km × 1þ þ½S × 1þ Kia Kib

Noncompetitive Same equation as above but this is a special case of mixed Ki ¼ IC50 ¼ inhibition where Kia Kib and Km remains unchanged.

aK ¼ ½E½I K ¼ ½ES½I ia ½EI (binding of inhibitor, I, to the enzyme, E); ib ½ESI (binding of the inhibitor, I, to the enzyme-substrate complex, ES). 1746 Haupt et al.

Fig. 1. Distribution of P450 enzymes and substrates evaluated in the determination of 343 Ki values for reversible P450 inhibition in human liver microsomes with 132 investigational drugs. Downloaded from used within a customized software program (DI IC50 LCMS Template version System (Thermo Scientific, Waltham, MA) with Crystal Reports-SAP 2.0.3) for Microsoft Excel Office 2000 (version 9.0; Microsoft Inc., Redmond, WA). Business Objects (SAP, Newtown Square, PA). The data (i.e., reaction rates Both software programs use a Levenberg-Marquardt algorithm (Levenberg, at all concentrations of inhibitor at all concentrations of P450 marker sub- 1944; Marquardt, 1963), also known as a damped least-squares algorithm, to fit strates) were fitted to the Michaelis-Menten equations for competitive, dmd.aspetjournals.org a nonlinear regression (sigmoidal) curve to IC50 data based on the following noncompetitive, uncompetitive, and mixed (competitive-noncompetitive) equation: inhibition (see Table 1) by nonlinear regression analysis. The goodness of fit to each of the four inhibition equations was determined by x2 analysis (with Max 2 Min ¼ þ ð Þ lower values indicating better fit) or by comparison of Akaike information Fit Min slope 3 ð Þ criterion values (with higher values indicating better fit), which provided an 1 þ Inhibitor IC50 initial basis for identifying the mechanism of inhibition. Eadie-Hofstee plots (rate versus rate/[S]) were inspected visually. At times, the nonlinear where Min = zero (no inhibition) and Max = 100 (complete inhibition). In regression lines did not correlate well with the data points depicted on the

XLfit, the terms Min and Max are called background and range, respectively. Eadie-Hofstee plot, and visual inspection of the kinetic plots was necessary to at ASPET Journals on September 28, 2021 Both software programs have been validated for their ability to calculate IC50 deduce the mechanism of inhibition. Both methods of data processing are values only when they lie within the actual range of inhibitor concentrations validatedtocalculateKi values only when they lie within the range of tested. In other words, none of the IC50 values reported here was extrapolated inhibitor concentrations tested. from data that fell above or below the highest or lowest concentration of Statistical Analysis. The accuracy of the prediction of observed Ki values inhibitor, respectively. from values of IC50/2 was assessed by determining the average fold error The data for Ki determinations were processed with one of two comparable (AFE) according to eq. 4 (Obach et al., 1997). This method is based on methods. The first method used Microsoft Excel to calculate rates of absolute values of the logarithm of the ratio of predicted-to-observed metabolite formation, which were imported into GraFit (Erithacus Software values, meaning that all negative values are converted to positive values so Ltd., Horley, Surrey, UK) to perform nonlinear regression according to the that, for example, values 50% less and 100% more than the observed value Michaelis-Menten equations associated with each type of direct inhibition. both represent a 2-fold error. An AFE value of 1 represents a perfect The second method used a Galileo Laboratory Information Management prediction.

TABLE 2

Distribution of the types of reversible inhibition observed for 343 Ki determinations for nine different P450 enzymes in pooled human liver microsomes

Mechanism of Inhibition P450 Enzyme Substrate Number Mixed Competitive Noncompetitive Uncompetitive CYP1A2 Phenacetin 21 (6%) 4 17 0 0 CYP2A6 Coumarin 6 (2%) 4 1 1 0 Efavirenz 8 (2%) 4 2 2 0 CYP2B6 Bupropion 14 (4%) 13 1 0 0 Amodiaquine 28 (8%) 24 4 0 0 CYP2C8 Paclitaxel 33 (10%) 26 2 5 0 CYP2C9 Diclofenac 47 (14%) 40 7 0 0 CYP2C19 S-mephenytoin 44 (13%) 35 9 0 0 Bufuralol 1 (,1%) 0 1 0 0 CYP2D6 Dextromethorphan 41 (12%) 22 19 0 0 CYP2E1 Chlorzoxazone 2 (1%) 2 0 0 0 Midazolam 57 (17%) 29 25 3 0 CYP3A4/5 Testosterone 31 (9%) 11 19 1 0 Nifedipine 10 (3%) 3 1 1 5 TOTAL 343 217 (63.3%) 108 (31.5%) 13 (3.8%) 5 (1.5%) Estimating Ki from IC50 Values for P450 Inhibition 1747

Fig. 2. Comparison of 343 observed (experimentally determined) Ki values with predicted Ki values from IC50/2 when [S] Km.The line shown is a line of identity. Downloaded from

1 Predicted Ki + log inhibitors of more than one P450 enzyme (data not shown). The n Observed K AFE ¼ 10 i ð4Þ correlations between the 343 observed (experimentally determined) Ki dmd.aspetjournals.org values and those predicted from the corresponding values of IC50/2 The precision of the prediction was assessed by calculating the root mean (when [S] Km) are shown in Fig. 2 and Table 3. The correlations square error (RMSE) according to eq. 5 (Sheiner and Beal, 1981): between observed and predicted Ki values as a function of the rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi mechanism of P450 inhibition are shown in Fig. 3. The ratios of the 1 RMSE ¼ + ðpredicted K 2 observed K Þ2 ð5Þ n i i observed-to-predicted Ki values as a function of the mechanism of P450 inhibition are shown in Fig. 4 and Table 3. Measures of the accuracy The normalized RMSE (NRMSE; expressed as a percentage) was calculated (AFE) and precision (RMSE and NRMSE) of predicting Ki values from as follows: at ASPET Journals on September 28, 2021 IC50/2 when [S] Km are shown in Figs. 2 and 3, and summarized in RMSE Table 3. NRMSE ¼ × 100 ð6Þ Max observed Ki 2 Min observed Ki Discussion In all cases, the observed Ki is the experimentally determined value, and the A total of 343 K values for reversible inhibition of various P450 predicted Ki corresponds to IC50/2. i enzymes was determined experimentally (from studies with multiple inhibitor concentrations versus multiple substrate concentrations) with Results 132 structurally diverse investigational drugs (some of which were The nine P450 enzymes and 14 marker substrates examined in the examined with more than one P450 enzyme) after determining IC50 current study, and the distribution of the types of reversible inhibition values in human liver microsomes under conditions where [S] Km. (i.e., competitive, noncompetitive, mixed, and uncompetitive inhibi- Table 2 shows the distribution of the four mechanisms of reversible tion) observed in vitro with human liver microsomes are summarized in inhibition for the 343 Ki determinations. The compounds were Fig. 1 and Table 2. The inhibitors were 132 structurally diverse predominantly (;95%) mixed (n = 217) or competitive (n = 108) investigational drugs, some of which were examined as reversible P450 inhibitors; however, noncompetitive inhibitors (n = 13) and

TABLE 3

Summary of the accuracy (AFE) and precision (RMSE and NRMSE) of predicting Ki values for reversible cytochrome P450 inhibition from values of IC50/2 when [S] Km

Ratio of Observed versus Predicted Ki Inhibition Type n Correlation coefficient (r) AFEa RMSEb NRMSEc Mean Range n , 0.5 n . 2.0

% Mixed 217 1.13 0.481–2.95 1 9 0.926 1.07 74.2 3.90 Competitive 108 1.19 0.335–3.41 1 9 0.985 1.10 35.8 2.57 Noncompetitive 13 1.90 1.26–2.79 0 6 0.994 1.85 79.2 17.2 Uncompetitive 5 1.35 0.725–2.55 0 1 0.951 1.24 3.85 33.5 Mixed, competitive, and uncompetitive 330 1.15 0.335–3.41 2 19 0.941 1.08 63.5 3.34 Total 343 1.18 0.335–3.41 2 25 0.940 1.10 64.2 3.38

aSee eq. 4. bSee eq. 5. cSee eq. 6. 1748 Haupt et al. Downloaded from dmd.aspetjournals.org

Fig. 3. Comparison of observed (experimentally determined) Ki values with Ki values estimated from values of IC50/2 (when [S] Km) as a function of the mechanism of reversible P450 inhibition. The line shown is a line of identity.

uncompetitive inhibitors (n = 5) were also represented in the data set. (based on IC50/2) correlated well (r = 0.940) with observed values of Ki, at ASPET Journals on September 28, 2021 The distribution of P450 enzymes analyzed as well as the marker and the prediction was both accurate (AFE = 1.10) and precise (RMSE = substrates used are shown in Fig. 1 and Table 2. For the seven drug- 64.2; NRMSE = 3.38%). metabolizing P450 enzymes listed in the FDA and EMA guidance When the data were segregated by inhibition type, the observed and documents on drug interactions, the number of Ki determinations predicted Ki values were highly correlated for all four types of inhibition ranged from 21 (CYP1A2) to 94 (CYP3A4/5). Relatively few Ki values (r values ranged from 0.926 to 0.994), as shown in Table 3 and Fig. 3. were determined with CYP2A6 (n = 6) and CYP2E1 (n = 2). Four P450 Values of IC50/2 served as accurate predictors of observed Ki values for enzymes (CYP2B6, 2C8, 2D6, and 3A4/5) were assayed with more competitive inhibition (AFE = 1.10) and uncompetitive inhibition (AFE = than one substrate. 1.24). This was as expected because, in theory, Ki should equal IC50/2 The correlation between the observed (experimentally determined) for these types of inhibition (Table 1). Values of IC50/2 also accurately Ki values and the predicted Ki values (from IC50/2) for the entire data set predicted Ki values for mixed inhibition (AFE = 1.07), where, in theory, is shown in Fig. 2. The experimentally determined Ki values ranged Ki values can range from IC50 to IC50/2 (Table 1). However, IC50/2 was 200,000-fold from 9 nM to 1.9 mM. Overall, predicted values of Ki a less accurate predictor of Ki values for noncompetitive inhibition. In this case, the AFE value of 1.85 indicates that the prediction was off by a factor of nearly 2, which is consistent with the equation in Table 1, indicating that, for noncompetitive inhibition, Ki =IC50, not IC50/2. Omitting the 13 Ki values for noncompetitive inhibition (where theoretically Ki =IC50 and not IC50/2) resulted in a negligible improvement in overall AFE; the value decreased from 1.10 to 1.08, and NRMSE decreased slightly from 3.38 to 3.34%, as shown in Table 3. The ratios of observed-to-predicted Ki values are shown in Fig. 4 and summarized in Table 3. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki value. Two predicted values were less than 0.5 (but not less than 0.33) and 25 values were greater than 2, only one of which was greater than 3. In other words, of the 343 predicted Ki values, 316 were within a factor of 2 and 342 were within a factor of 3, meaning only one value fell outside a 3-fold range (the actual value was 3.41). A preliminary account of this work included a second case in which the predicted Ki value was more Fig. 4. Ratio of observed (experimentally determined) Ki values with Ki values estimated from values of IC50/2 (when [S] Km) as a function of the mechanism of than 3-fold greater than the experimentally determined Ki value (Haupt reversible P450 inhibition. The line shown is a line of identity. et al., 2011). However, in this case, the initial IC50 value was below the Estimating Ki from IC50 Values for P450 Inhibition 1749 lowest concentration of inhibitor tested, and an interpolated IC50 value Overall, our analysis of 343 determinations supports theoretical was used in the analysis. This observation underscores the importance considerations (Table 1) that, when determined under in vitro of predicting Ki values only when the IC50 value falls within the range conditions of low protein concentration, short substrate incubation of inhibitor concentrations tested. time, and [S] Km, values of IC50/2 provide an accurate prediction It is important to note that other experimental conditions can affect (generally within a factor of 2) of experimentally determined Ki values the measured values of IC50 and the kinetic constants Km and Ki (Obach, for all types of reversible, direct inhibition of P450 enzymes. In the case 1996, 1997, and 1999; McLure et al., 2000; Austin et al., 2002; Di of noncompetitive inhibitors, values of Ki estimated from IC50/2 were Marco et al., 2003; Margolis and Obach, 2003; Hallifax and Houston, off by a factor of ;2 (AFE = 1.85), which is consistent with theoretical 2006; Howgate et al., 2006; Brown et al., 2007a,b; Ogilvie et al., 2011; considerations (Table 1). However, noncompetitive inhibitors Parkinson et al., 2011). In this study, all Ki and IC50 values were accounted for a relatively low percentage (;4%) of the types of determined at 0.1 mg microsomal protein/ml or less with a short inhibition observed (13 of 343 determinations). In conclusion, the substrate incubation time (5 minutes) to minimize metabolic loss of the results of our analysis suggest that, under appropriate experimental inhibitory drug, to reduce the possibility of metabolism-dependent conditions, Ki values for direct, reversible inhibition can be reliably, inhibition, and to maximize the concentration of unbound inhibitor. albeit somewhat conservatively, estimated from values of IC50/2. Membrane partitioning [commonly but erroneously called nonspecific The following excerpt is from the FDA’s Guidance for Industry on binding (Nagar and Korzekwa, 2012)] decreases the unbound concen- drug interactions (from footnote 2 on page 21): “For a drug that is tration of inhibitor, but this effect can be corrected by measuring or a reversible inhibitor, R = 1+[I]/Ki.Ki is the unbound inhibition Downloaded from calculating fuinc (Austin et al., 2002; Hallifax and Houston, 2006) and constant determined in vitro. Sometimes inhibitor concentration expressing the inhibition constant as Ki,unbound, now recommended by causing 50% inhibition (IC50) is determined, and Ki can be calculated both the FDA and EMA. This same approach can be applied to as IC50/2 by assuming competitive inhibition.” The results of our measurements of Ki based on IC50/2. It should be noted that, in the analysis suggest that measuring IC50 is sufficient to estimate Ki for current study, IC50 and Ki values for a given P450 enzyme were the purpose of evaluating the potential of an investigational drug to determined under identical experimental conditions (i.e., the same cause clinically relevant P450 inhibition, with two stipulations. First, dmd.aspetjournals.org concentration of microsomal protein), such that values of fuinc were the IC50 is determined under appropriate experimental conditions, as same for predicted and observed Ki determinations. Accordingly, described in the previous paragraph. Second, the Ki value estimated correcting for fuinc would have had no effect on estimates of the ratio from IC50/2 is corrected for the fraction of unbound drug in the test of the prediction-to-observed Ki values or the accuracy and precision of system, such that Ki,u is estimated from (IC50/2)×fuinc, where fuinc is predicting Ki values from IC50/2. determined experimentally or calculated from log P (in the case of Although the experimentally determined Ki values varied approxi- nonionic and basic drugs) or log D7.4 (in the case of acidic and mately 200,000-fold, most of them (240 of 343) fell between 1 and zwitterionic drugs), as described by Austin et al. (2002) and Hallifax

25 mM (with 61 greater than 25 mM and 42 less than 1 mM). In the majority and Houston (2006). at ASPET Journals on September 28, 2021 of cases (287 of 343 determinations), the initial IC50 value was 50 mM Acknowledgments or less (hence, the predicted Ki value was 25 mM or less), suggesting that the decision to measure Ki values was biased toward those cases in The authors thank the Analytical Sciences and Enzyme Inhibition depart- ments at XenoTech LLC for technical assistance. which the initial IC50 determination suggested relatively strong P450 inhibition. There were 37 cases in which the initial IC50 was greater than 100 mM (i.e., the inhibition was relatively weak). It might be expected Authorship Contributions Participated in research design: Kazmi, Ogilvie, Buckley, Paris, that, in these cases, the decision to perform a Ki determination was biased toward CYP3A4/5 because of its intestinal location, where A. Parkinson. Conducted experiments: Haupt, Smith, Leatherman. the relevant inhibitor concentration is molar dose/250 ml, not plasma Performed data analysis: Haupt, Kazmi, Ogilvie, Buckley, Paris, Cmax,ss. However, of the 37 cases of weak inhibition, Ki values were O. Parkinson, A. Parkinson. determined for a wide variety of P450 enzymes: 13 for CYP3A4/5, 6 Wrote or contributed to the writing of the manuscript: Haupt, Kazmi, for CYP2C9, 6 for CYP2C19, 5 for CYP2D6, 3 for CYP2B6, 2 for Ogilvie, Buckley, O. Parkinson, Paris, A. Parkinson. CYP1A2, 1 for CYP2A6, and 1 for CYP2C8. Therefore, CYP3A4/5 represented only 35% of the cases in which Ki was determined when the References m initial IC50 was greater than 100 M. Austin RP, Barton P, Cockroft SL, Wenlock MC, and Riley RJ (2002) The influence of non- Interestingly, uncompetitive inhibition, characterized by a decrease specific microsomal binding on apparent intrinsic clearance, and its prediction from physico- in both V and K , was observed only when nifedipine was the chemical properties. Drug Metab Dispos 30:1497–1503. max m Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, Kao J, King SP, Miwa G, substrate. Of the 10 assays performed with nifedipine, 5 exhibited and Ni L, et al.; Pharmaceutical Research and Manufacturers of America (PhRMA) Drug uncompetitive inhibition. The CYP3A substrates used in the current Metabolism/Clinical Pharmacology Technical Working Group; ; FDA Center for Drug Eval- uation and Research (CDER) (2003) The conduct of in vitro and in vivo drug-drug interaction study, midazolam, testosterone, and nifedipine, are thought to bind to studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug three distinct regions within the substrate-binding site and exhibit Metab Dispos 31:815–832. Brandt RB, Laux JE, and Yates SW (1987) Calculation of inhibitor Ki and inhibitor type from the distinct enzyme kinetics characterized by hyperbolic (typical concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes. Biochem Med Michaelis-Menten) kinetics in the case of midazolam, substrate Metab Biol 37:344–349. Brown HS, Chadwick A, and Houston JB (2007a) Use of isolated hepatocyte preparations for activation (sigmoidal kinetics due to homotropic cooperativity) in the cytochrome P450 inhibition studies: comparison with microsomes for Ki determination. Drug case of testosterone, and substrate inhibition in the case of nifedipine. Metab Dispos 35:2119–2126. Brown HS, Griffin M, and Houston JB (2007b) Evaluation of cryopreserved human hepatocytes The high prevalence of uncompetitive inhibition observed with as an alternative in vitro system to microsomes for the prediction of metabolic clearance. Drug nifedipine, which suggests some inhibitors bind to the CYP3A- Metab Dispos 35:293–301. Cer RZ, Mudunuri U, Stephens R, and Lebeda FJ (2009) IC50-to-Ki: a web-based tool for nifedipine (enzyme-substrate) complex, is consistent with previous converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding. Nucleic studies documenting the so-called stand-alone properties of nifedipine Acids Res 37:W441–W445. Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the as a CYP3A4/5 substrate (Kenworthy et al., 1999; Wang et al., 2000; concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Galetin et al., 2002, 2003; Foti et al., 2010). Biochem Pharmacol 22:3099–3108. 1750 Haupt et al.

Di Marco A, Yao D, and Laufer R (2003) Demethylation of radiolabelled dextromethorphan in rat Obach RS (1999) Prediction of human clearance of twenty-nine drugs from hepatic microsomal microsomes and intact hepatocytes. Eur J Biochem 270:3768–3777. intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding Foti RS, Rock DA, Wienkers LC, and Wahlstrom JL (2010) Selection of alternative CYP3A4 to microsomes. Drug Metab Dispos 27:1350–1359. probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation. Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ, and Wastall P Drug Metab Dispos 38:981–987. (1997) The prediction of human pharmacokinetic parameters from preclinical and in vitro Galetin A, Clarke SE, and Houston JB (2002) Quinidine and haloperidol as modifiers of CYP3A4 metabolism data. J Pharmacol Exp Ther 283:46–58. activity: multisite kinetic model approach. Drug Metab Dispos 30:1512–1522. Ogilvie BW, Yerino P, Kazmi F, Buckley DB, Rostami-Hodjegan A, Paris BL, Toren P, Galetin A, Clarke SE, and Houston JB (2003) Multisite kinetic analysis of interactions between and Parkinson A (2011) The proton pump inhibitor, omeprazole, but not lansoprazole or prototypical CYP3A4 subgroup substrates: midazolam, testosterone, and nifedipine. Drug pantoprazole, is a metabolism-dependent inhibitor of CYP2C19: implications for co- Metab Dispos 31:1108–1116. administration with clopidogrel. Drug Metab Dispos 39:2020–2033. Hallifax D and Houston JB (2006) Binding of drugs to hepatic microsomes: comment and Paris BL, Ogilvie BW, Scheinkoenig JA, Ndikum-Moffor F, Gibson R, and Parkinson A (2009) assessment of current prediction methodology with recommendation for improvement. Drug In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran. Metab Dispos 34:724–726, author reply 727. Drug Metab Dispos 37:2045–2054. Haupt L, Kazmi K, Ogilvie B, Buckley D, Smith B, Leatherman S, and Parkinson A (2011) Can Parkinson A, Kazmi F, Buckley DB, Yerino P, Paris BL, Holsapple J, Toren P, Otradovec Ki values for direct inhibition of CYP enzymes be reliably estimated from IC50 values? Drug SM, and Ogilvie BW (2011) An evaluation of the dilution method for identifying Metab Rev 43 (S2):105. metabolism-dependent inhibitors of cytochrome P450 enzymes. Drug Metab Dispos 39: Howgate EM, Rowland Yeo K, Proctor NJ, Tucker GT, and Rostami-Hodjegan A (2006) Pre- 1370–1387. diction of in vivo drug clearance from in vitro data. I: impact of inter-individual variability. Parkinson A, Mudra DR, Johnson C, Dwyer A, and Carroll KM (2004) The effects of gender, Xenobiotica 36:473–497. age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H, and Sugiyama Y (1998) Prediction of microsomes and inducibility in cultured human hepatocytes. Toxicol Appl Pharmacol 199: pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. 193–209. Pharmacol Rev 50:387–412. Pearce RE, McIntyre CJ, Madan A, Sanzgiri U, Draper AJ, Bullock PL, Cook DC, Burton LA, Kenworthy KE, Bloomer JC, Clarke SE, and Houston JB (1999) CYP3A4 drug interactions: Latham J, and Nevins C, et al. (1996) Effects of freezing, thawing, and storing human liver correlation of 10 in vitro probe substrates. Br J Clin Pharmacol 48:716–727. microsomes on cytochrome P450 activity. Arch Biochem Biophys 331:145–169. Levenberg K (1944) A Method for the Solution of Certain Non-Linear Problems in Least Squares. Rodrigues AD, Winchell GA, and Dobrinska MR (2001) Use of in vitro drug metabolism data to Downloaded from Q Appl Math 2:164–168. evaluate metabolic drug-drug interactions in man: the need for quantitative databases. J Clin Margolis JM and Obach RS (2003) Impact of nonspecific binding to microsomes and phos- Pharmacol 41:368–373. pholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition Sheiner LB and Beal SL (1981) Some suggestions for measuring predictive performance. J data to in vivo drug interactions. Drug Metab Dispos 31:606–611. Pharmacokinet Biopharm 9:503–512. Marquardt D (1963) An Algorithm for Least-Squares Estimation of Nonlinear Parameters. SIAM J Tucker GT, Houston JB, and Huang SM (2001) Optimizing drug development: strategies to Appl Math 11:431–441. assess drug metabolism/transporter interaction potential–toward a consensus. Pharm Res 18: McLure JA, Miners JO, and Birkett DJ (2000) Nonspecific binding of drugs to human liver 1071–1080. microsomes. Br J Clin Pharmacol 49:453–461. Wang RW, Newton DJ, Liu N, Atkins WM, and Lu AY (2000) Human cytochrome P-450 3A4: Nagar S and Korzekwa K (2012) Commentary: nonspecific protein binding versus membrane in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab Dispos 28: dmd.aspetjournals.org partitioning: it is not just semantics. Drug Metab Dispos 40:1649–1652. 360–366. Obach RS (1996) The importance of nonspecific binding in in vitro matrices, its impact on enzyme kinetic studies of drug metabolism reactions, and implications for in vitro-in vivo correlations. Drug Metab Dispos 24:1047–1049. Address correspondence to: Obach RS (1997) Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic Dr. Andrew Parkinson, XPD Consulting, 18000 clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and West 68th Street, Shawnee, KS 66217. E-mail: [email protected] propranolol. Drug Metab Dispos 25:1359–1369. at ASPET Journals on September 28, 2021 DMD #66597

Supplemental Table

The reliability of estimating Ki values for direct, reversible inhibition of cytochrome P450 enzymes from corresponding IC50 values: A retrospective analysis of 343 experiments

Lois J. Haupt, Faraz Kazmi, Brian W. Ogilvie, David B. Buckley, Brian D. Smith, Sarah Leatherman, Brandy Paris, Oliver Parkinson, and Andrew Parkinson

XenoTech, LLC, Lenexa, KS, USA (L.J.H., F.K., B.W.O., D.B.B., B.D.S., S.L.) XPD Consulting, Shawnee, KS, USA (B.P., O.P., A.P.)

Drug Metabolism and Disposition

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DMD #66597

Supplemental Table 1: Summary of experimental conditions for measuring cytochrome P450 inhibition in human liver microsomes (HLM)

P450 P450 Enzyme [Substrate]2 [Protein] Analyte measured by LC-MS/MS 1 Enzyme Activity (Km) (mg/mL) (internal standard)

Phenacetin Acetaminophen CYP1A2 40 – 60 or 90 µM 0.1 O-dealkylation (Acetaminophen-d4)

Coumarin 7-Hydroxycoumarin CYP2A6 0.6 - 0.75 µM 0.0125 or 0.1 3 7-hydroxylation (7-Hydroxycoumarin-d5)

Bupropion Hydroxybupropion 50 µM 0.1 hydroxylation (Hydroxybupropion-d6) CYP2B6 Efavirenz 8-Hydroxyefavirenz 3 or 5 µM 0.1 8-hydroxylation (8-Hydroxyefavirenz-d4)

Amodiaquine N-Desethylamodiaquine 1.5 – 7 or 2 µM 0.0125 or 0.1 3,4 N-dealkylation (N-Desethylamodiaquine-d5) CYP2C8 6α-Hydroxypaclitaxel Paclitaxel 10 µM 0.05 (6α-Hydroxypaclitaxel-d5 or 6α-hydroxylation deacetyltaxol) Diclofenac 4′-Hydroxydiclofenac CYP2C9 6 – 7.5 or 12 µM 0.1 4′-hydroxylation (4′-Hydroxydiclofenac-d4)

S-Mephenytoin 4′-Hydroxymephenytoin CYP2C19 40 or 60 µM 0.1 4′-hydroxylation (4′-Hydroxymephenytoin-d3)

Dextromethorphan Dextrorphan 7.5 or 10 µM 0.1 O-demethylation (Dextrorphan-d3) CYP2D6 Bufurolol 1′-Hydroxybufurolol 12 µM 0.1 1′-hydroxylation (Dextrorphan-d3)

Chlorzoxazone 6-Hydroxychlorzoxazone CYP2E1 30 µM 0.1 6-hydroxylation (6-Hydroxychlorzoxazone-d2)

Testosterone 5 6β-Hydroxytestosterone 70-100 or 60 µM 0.1 6β-hydroxylation (6β-Hydroxytestosterone-d3)

Midazolam 1′-Hydroxymidazolam CYP3A4/5 2 - 5 or 3 µM 0.05 or 0.1 3 1′-hydroxylation (1′-Hydroxymidazolam-d4) Nifedipine Dehydronifedipine 10 µM 0.1 dehydrogenation (Dehydronifedipine-d6)

1 All substrates were incubated for 5 min. 2 Substrate concentrations in plain text are for the HLM pool of 16 whereas values in italics are for the HLM pool of 200. 3 Active microsomal protein concentration was normalized to 0.1 mg/mL with an individual preparation of HLM with low enzyme activity. 4 A single assay was performed with 0.1 mg/mL of active HLM and a marker substrate concentration of 7 µM. 5 In the case of testosterone, which exhibits homotropic cooperativity (substrate activation), the value represents S50 (with a Hill coefficient of n = 1.3).

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